Although several studies suggest that myocardial injury during ischemia/reperfusion is in part due to reactive oxygen species, the mechanisms by which these oxidants injure the heart and those by which the heart protects itself from their effects are not known. We propose that the myocardial effects of oxidative stress are mediated in part by 4-hydroxyalkenals such as 4-hydroxy-trans-2-nonenal (HNE), which are derived from oxidation of lipoproteins and membrane lipids. Our previous work has led to the identification of the major biochemical pathways of HNE detoxification in cardiovascular tissues. These results suggest that aldose reductase (AR)-catalyzed reduction of HNE and its glutathione conjugate is a particularly critical step in this metabolism because inhibition of AR increases HNE accumulation in the ischemic heart and abolishes the cardioprotective effects associated with the late phase of ischemic preconditioning (PC). Based on these observations and our preliminary data, we now plan to test how the ischemic heart metabolizes HNE and whether this is facilitated by the activation of AR due to nitric oxide (NO) generated during ischemia. Using both ex vivo and in situ murine models of myocardial ischemia-reperfusion injury, we will identify ischemic changes in AR and HNE metabolism and identify the chemical nature of AR modification and the oxidants and triggers that induce it (Aim 1). In addition, we will determine the role of endothelial and inducible NO synthases (NOS) in activating AR and increasing HNE detoxification in the ischemic heart using mice deficient in individual NOS isoforms or overexpressing inducible NO synthase in the heart (Aim 2). To understand the mechanisms of AR-mediated cardioprotection, we will examine changes in the mitochondria and myocardial survival signaling (Aim 3) and elucidate how pharmacologic inhibition of AR or cardiospecific overexpression of AR (or its murine homolog FR-1) affects ischemia-reperfusion-induced mitochondrial permeability transition and NF-kappaB activation (Aim 3). Successful completion of this project will lead to a better understanding of mechanisms of cardioprotection due to ischemic PC and NO and could form the basis of new clinical approaches to limit myocardial infarction. Delineation of the contribution of specific enzymes to HNE metabolism in the ischemic heart will be useful in identifying population subgroups that may be highly susceptible to oxidative stress caused by cardiovascular disease.